The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Synthetic oligonucleotides tethered to various ligands have been used as research tools in molecular biology [see e.g.: Goodchild, Bioconjugate Chem., 1990, 3, 166; Uhlman and Peyrnan, Chem. Rev., 1990, 90, 543; Sigman, et al. Chem. Rev., 1993, 93, 2295; O'Donnel and McLaughlin in Bioorganic Chemistry, Nucleic Acids, Hecht S M, ed. Oxford Univ. Press, 1996, p. 216]. They have been applied to genetic analysis, and to elucidate mechanism of gene function. Oligonucleotides carrying reporter groups have had widespread use for automated DNA sequencing, hybridization affinity chromatography and fluorescence microscopy. Oligonucleotide-biotin conjugates are widely used as hybridization probes. Antisense oligonucleotides covalently linked to intercalators, chain cleaving or alkylating agents have been shown to be efficient as gene expression regulators. The sequence specific artificial nucleases, when targeted against mRNA, may find applications even as chemotherapeutics.
For several applications, such as in DNA hybridization assays, it is desirable to introduce more than one reporter group to the oligonucleotide structure. This can be performed by three alternative methods:                (i) by coupling several base- or carbohydrate-tethered nucleosidic building blocks to the growing oligonucleotide chain,        (ii) by functionalization of the internucleosidic phosphodiester linkages, or        (iii) by using several multifunctional non-nucleosidic building blocks during the oligonucleotide chain assembly.        
All of these methods have their own drawbacks. Since the double helix formation of DNA is based on hydrogen bonding between the complementary base residues, tethers attached to the base moieties often weaken these interactions. This problem is easily overcome by using the tethered nucleosides at the 3′- or 5′-terminus of the coding sequence, or by using labels linked to C5 of pyrimidine residues. Introduction of tethers to the phosphate backbone gives rise to new chiral centers and makes the purification of these analogues difficult. Introduction of the tether arm to the carbohydrate moiety, in turn, often decreases the coupling efficiency of the phosphoramidite (steric hindrance). Furthermore, synthesis of these blocks is commonly extremely laborious. Although design of non-nucleosidic blocks may look attractive on paper, very often their syntheses suffer from complexity, low coupling yields and problems associated with the storage and handling of the phosphoramidites. For commercial applications design of base tethered nucleosidic building blocks is often the method of choice.
Introduction of linker arms to the nucleobase is most commonly performed by allowing a nucleoside with a good leaving group (N-tosyl, N-benzoyl, halogen, triazole, thiol) at C4 of pyrimidines or C2, C8 or C6 of purines to react with the appropriate nucleophilic linker molecule (e.g. an alkane-α,ω-diamine). Since normally an excess of linker molecule and rather vigorous reaction conditions has to be used, laborious purification procedures cannot be avoided. The basic reaction conditions needed gives additional requirements to the protecting groups in the target molecule. These problems may be overcome by attachment of the linker molecules to C5 of pyrimidine bases by a palladium catalyzed coupling reaction between 5-halogeno pyrimidine 5-mercuriochloro nucleoside and an alkynyl or allyl linker, respectively. However, the method involves rather laborious synthesis of a 5-halogeno or 5-mercuriochloro nucleoside. Very recently, attachment of a linker arm to the N3 of 3′,5′-O -protected thymidine based on Mitsunobu reaction [Mitsunobu, Synthesis, 1981, 1] was reported [J. Org. Chem., 1999, 64, 5083; Nucleosides, Nucleotides, 1999, 18, 1339]. Since the coupling reaction is performed under mild conditions, a wide range of tether arms can be introduced.
Most of the methods for oligonucleotide tethering described in literature involves attachment of functional groups in the oligonucleotide structure during chain assembly. Hence, introduction of the label molecules has to be performed in solution. In the labeling reaction the additional amino or mercapto groups of oligonucleotides are allowed to react in solution with isothiocyanato, haloacetyl or 2,4,6-triazinyl derivatives of label molecules. Carboxylic acid groups, in turn, can be labeled with amino tethered labels with the aid of water-soluble carbodiimide. Since in all the cases the labeling reaction is performed in aqueous solution with an excess of labeling reactants, laborious purification procedures cannot be avoided. Especially when attachment of several labels is required the isolation and characterization of the desired conjugate is extremely difficult, and often practically impossible. Hence, several attempts to incorporate label molecules or their appropriately protected precursor to oligonucleotide structure during chain assembly have been done [Ruth, J L et al, U.S. Pat. No. 4,948,882; Brush, C K et al, U.S. Pat. No. 5,583,236]. The fluorescent label monomers for solid phase chemistry synthesized are most commonly organic dyes (e.g. fluorescein, rhodamine, dansyl, dabsyl, pyrene, TAMRA) several of these are even commercially available. However, such labels and labeled biomolecules suffer from many commonly known drawbacks such as Raman scattering, other fluorescent impurities, low water solubility, concentration quenching etc. In the specific binding assays, generally very low concentrations of analytes to be measured are present. Thus multilabeling of oligonucleotides with organic fluorophores may not enough enhance detection sensitivity needed in several applications. For these types of applications lanthanide(III) chelates are labels of choice since they do not suffer from this phenomenon. In DNA hybridization assays, time-resolved luminescence spectroscopy using lanthanide chelates is well known [Hemmilä et al. Bioanalytical Applications of Labelling Technologies, Wallac Oy, 1994]. Therefore, a number of attempts have been made to develop non-luminescent (DELFIA®) and new highly luminescent chelate labels suitable for time-resolved fluorometric applications. Many patent publications disclose non-luminescent labels [e.g. EP 0064484 A2, EP 0139675 B1, EP 0298939 A1, U.S. Pat. Nos. 4,808,541 and 4,565,790]. Highly luminescent labels include e.g. stabile chelates composed of derivatives of pyridines [U.S. Pat. Nos. 4,920,195, 4,801,722, 4,761,481, WO 93/11433, U.S. Pat. No. 4,459,186, EP 0770610 A1 and Remuinan et al, J. Chem. Soc. Perkin Trans 2, 1993, 1099], bipyridines [U.S. Pat. No. 5,216,134], terpyridines [U.S. Pat. Nos. 4,859,777, 5,202,423 and 5,324,825] or various phenolic compounds [U.S. Pat. Nos. 4,670,572, 4,794,191 and Ital. Pat. 42508 A789] as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives [U.S. Pat. Nos. 5,032,677, 5,055,578 and 4,772,563] macrocyclic cryptates [U.S. Pat. No. 4,927,923, WO 93/5049 and EP 0493745 A1] and macrocyclic Schiff bases [EP 369000 A] have been patented. Also a method for labeling of biospecific binding reactants such as hapten, a peptide, a receptor ligand, a drug or PNA oligomer with luminescent labels by using solid-phase synthesis has been published [EP 067205A1]. One such oligonucleotide labeling reagent has been synthesized and used in multilabeling of oligonucleotides [Kwiatkowski et al. Nucleic Acids Res., 22, 1994, 2604]. However the synthetic strategy described allows only preparation of chelates where the nucleobase is conjugated to the chelate structure limiting the chelate stability and versatility. Furthermore, the structure synthesized is usable only with europium(III) but not with terbium(III), dysprosium(III) or samarium(III).
For some special applications such as helicase assays based on fluorescence energy transfer [Earnshaw et. al, J. Biomol. Screening, 4, 1999, 239] large quantities of ultrapure oligonucleotides bearing a luminescent lanthanide(III) chelate at their 3′- or 5′-terminus are needed. Although these molecules can be obtained by classical labeling methods in solution, yields of the oligonucleotide conjugates can be dramatically improved and purification procedures can be highly simplified if the label could be attached to the oligonucleotide structure during chain assembly. For 5′-derivatization synthesis of nucleosidic or non-nucleosidic building blocks are needed, while 3′-labeling calls for appropriately derivatized polymeric solid supports.